Volatile anesthetics perturb the coupling of metabotropic receptors to specific heterotrimeric guanine nucleotide-binding proteins (G-proteins), but the relevance to anesthesia and its molecular basis are not understood. Heterotrimeric G-proteins, consisting of alpha, beta, and gamma subunits, are GTP-driven molecular switches that couple metabotropic receptors to downstream effectors, including ion channels. In the inactive state, the G-protein is a heterotrimer with GDP tightly bound to the Galpha subunit. Binding of agonist to the receptor triggers exchange of GTP for bound GDP, hydrolyzing the former in the presence of Mg2+. The GTP-charged alpha subunit dissociates from betagamma and may be free to diffuse some distance on the membrane surface. Either the alpha subunit bound to GTP or the free betagamma heterodimer or both regulate effector enzymes such adenyl cyclase (AC) and phospholipase C, or modulate ion channels, thereby amplifying the initial receptor stimulus. Thus, alpha subunits function as on/off switches based on the occupant of the nucleotide binding site, GTP or GDP, such that alterations in nucleotide exchange, like those induced by volatile anesthetics, modulate signal output. To test for correlation between clinical potency and inhibition of GDP/GTP exchange, various anesthetic compounds, as well as non-immobilizing drugs, will be tested on Galpha subunits. Similar experiments will be performed on living cells in which the pathways are reconstructed by expression of the signaling components: receptors, G-protein subunits and effectors. The hypothesis that volatile agents disrupt receptor/effector coupling by locking the Galpha subunit into a complex with Gbetagamma will be tested on artificial membranes by measuring the lateral interactions of these proteins using fluorescence resonance energy transfer. To understand the molecular basis of the anesthetic effect, intrinsic fluorescence and isothermal titration microcalorimetry will be used to measure the binding of these drugs to soluble Galpha subunits. Finally, regions essential to the actions of anesthetics will be established by swapping homologous portions of sensitive and insensitive alpha subunits. These studies will generate predictions for future experiments to be conducted on animals exposed to anesthetic agents. Completion of the proposed study will also form the basis for future crystallographic work on the nature of interactions between anesthetics and these potential protein targets.